Microbial production of natural sweeteners, diterpenoid steviol glycosides

1. A method for producing steviol or steviol glycoside comprising:

culturing an E. coli strain having balanced expression of (1) an upstream methylerythritol pathway (MEP) that produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), with respect to (2) a downstream pathway that produces steviol or steviol glycoside from said IPP and DMAPP, the downstream pathway comprising a recombinantly expressed copalyl diphosphate synthase (CPS), kaurene synthase (KS), a geranylgeranyl diphosphate synthase (GGPPS) kaurenoic acid 13-hydroxylase (KAH) and kaurene oxidase (KO), and optionally one or more Stevia UDP glycosyl transferase enzymes;

wherein said balanced expression is obtained by increasing or decreasing the expression level of a downstream pathway module and increasing or decreasing the expression level of an upstream pathway module together in E. coli , and identifying an E. coli strain with higher production of steviol or steviol glycoside and/or lower accumulation of indole as having balanced expression.

2. The method of claim 1 , wherein the copalyl diphosphate synthase (CPS) enzyme is a Stevia enzyme.

3. The method of claim 1 , wherein the kaurene synthase (KS) enzyme is a Stevia enzyme.

4. The method of claim 1 , wherein the GGPPS enzyme is a Taxus enzyme or a Stevia enzyme.

5. The method of claim 1 , wherein the upstream pathway module comprises dxs, idi, ispD, and ispF genes of the MEP pathway.

6. The method of claim 5 , wherein the upstream pathway module comprises dxs, idi, ispD and ispF genes of the MEP pathway expressed as the operon dxs-idi-ispD-ispF.

7. The method of claim 1 , wherein the downstream module comprises the gene encoding the copalyl diphosphate synthase (CPS) enzyme, the gene encoding the kaurene synthase (KS) enzyme and the gene encoding the GGPPS enzyme co-expressed on an operon.

8. The method of claim 1 , wherein the downstream module further comprises kaurene oxidase (KO) and kaurenoic acid 13-hydroxylase (KAH) enzymes co-expressed on an operon, optionally each as fusions with a cytochrome P450 reductase.

9. The method of claim 1 , wherein the expression of the upstream pathway module and the expression of the downstream pathway module are balanced by one or more of: increasing or decreasing promoter strengths, increasing or decreasing gene or operon copy number, and changing the position of genes within the module.

10. The method of claim 9 , wherein one or more operons is integrated into the E. coli genome.

11. The method of claim 1 , wherein the KAH and KO are Stevia enzymes.

12. The method of claim 11 , wherein the KAH and/or KO comprise catalytically active portions fused to a Stevia cytochrome P450 reductase enzyme.

13. The method of claim 12 , wherein the KAH and KO enzymes have an N-terminal truncation and contain the N-terminal peptide sequence MALLLAVF (SEQ ID NO: 16).

14. The method of claim 1 , further comprising recovering the steviol or steviol glycoside.

15. The method of claim 14 , wherein the steviol or steviol glycoside is recovered from the gas phase of the culture by adding an organic layer.